Identification Of Genes Related To Volatile Terpenoids Biosynthesis In Chimonanthus Praecox Based On RNA-seq Technology

Wintersweet(Chimonanthus praecox)is a traditional ornamental plant endemic in China being cultivated for over 1000 years and prevalent for its rich floral scent.In our study,RNA seq and DGE of wintersweet was constracted by using Illumina paried-end sequencing technology.With the results of annotation and expression pattern,candidate genes in the pathway related to terpenoids biosynthases including MVA and MEP pathway genes,PTS genes,TPS genes and TF genes were analyzed and discussed.cNDA Full length of 2 CpTPSs were obtained by RACE approach,function annotations were operated via tobaco transformation.The main results were as follows:1.RNA-seq of 2 distinguishing genotypes10 samples from different stages of 2 distinguishing genotypes H29 and H64,were collected for RNA-seq using Illumina paried-end sequencing technology.3.5G and 4.8G data were obtained for this 2 transcriptome respectively and they were assembled into 89200 and 73952 Unigenes with an averange length of 565 bp and 648 bp.N50 were 869 and 1169.8.3 billion clean nucleotides from the integrated library were assembled into 83257 Unigenes.50213(60.31%)Unigenes showed similarity to known proteins,12576 Unigenes had significant hit among all the databases.2.Candidate genes from pathwaywith the help of function annotations of Unigenes,101 terpenoids synthase genes(including 24 mevalonic acid pathway genes and 37 methylerythritol phosphate pathway genes),16 prenyltransferase genes,24 terpene synthase genes were isolated from the transcriptome library.There were 1656 transcription factors were discovered as well,which belong to 51 transcription factor families of vascular plants,including MYB,bHLH,WRKY and AP2/ERF etc..3.Digital gene expression(DGE)profilesTo characterize the DGE profiles of wintersweet,10 DGE libraries were constructed and sequenced using paried-end sequencing technology to reveal the key enzyme genes involved in floral scents emission.Over 3 million row tags were obtained for each sample,95%of the raw tags passed the filter resulting clean tags.80%clean tags were mapped to the transcripts,and distinct tags arranged from 84.97%to 78.49%.After performing the annotation of assembles,Unigene involved in developmental changes were mapped onto glycolysis and the pentose phosphate(PPP),methylerythritol phosphate and mevalonate pathway with the number of 39,10 and 19.4.Different expression gene analysisWith the results of edgeR analysis,pathway different expression gene analysis and the co-expression gene selection,7 pathway Unigenes,8 up-regulated and 2 down-regulated Unigenes interested us thus the qRT-PCR was then performed to reveal:a)the expression profiles in small bud,initiation period and full-open samples in 2 genotypes;b)the expression profiles of 10 genotypes;c)the expression profiles of 5 different organs;d)the accuracy of DEG sequencing;e)effects of cold and dark treatment.5.Gene cloning of CpTPS313 and CpTPS712By RT-PCR and RACE,we isolated CpTPS313 and CpTPS172 gene from wintersweet flower.The full-length sequence of cDNA contained a total of 1629 and 1972 nucleotides,respectively,and encoded an ORF of 1512 and 1758 nucleotides,corresponding to a protein of 504 and 586 amino acids.Sequence analysis shows that CpTPS313 closely related to linalool synthase,and CpTPS172 closely related to alpha-terpene synthase or ocimene synthase.Both of them exhibits the features of terpene-synthase,belonging to terpene-synthase family.6.Function annotation of CpTPS313 and CpTPS712 via transgenetic tobaccoThe method of HS-SPME-GC-MS was applied to determine the volatile compounds in flowers of transgenetic tobacco to identify the function annotation of CpTPS313 and CpTPS172.It showed that,only tobacco-CpTPS172 were detected new compounds,the majority VOC were diterpene and sesquiterpene derivatives.We can conjecture that CpTPS172 is multi-functional and can be catalyzed the formation of terpenoids using both FPP and GGPP as substrate.This study set out to obtained differentially expressed genes for isolating key genes related to terpenoids VOC via constructing transcriptome and DGE libraries of wintersweet flowers,and also give insight to investigate the mechanism of terpenoids VOC biosynthesis and emission.